The present invention relates to isolated animal soluble adenylyl cyclase and its role in the regulation of the cAMP signaling pathway.
Adenylyl cyclase (AC) is the effector molecule of one of the most widely used signal transduction pathways. Its product, cyclic AMP (cAMP), is a nearly universally utilized second messenger molecule, which mediates cellular responses to nutritional conditions and extracellular signals in organisms from prokaryotes to higher eukaryotes. cAMP has long been known to exert both stimulatory and inhibitory effects on cell growth and proliferation (Dumont, J. E., et al., Trends Biochem. Sci., 1989, 14:67-71; Rozengurt, E., Science, 1986, 234:161-6). In metazoans, a seemingly ubiquitous membrane-associated AC activity is encoded by a family of transmembrane adenylyl cyclases (tmACs) that mediate cellular responses to external stimuli.
Throughout the animal kingdom members of the transmembrane adenylyl cyclase (tmAC) superfamily synthesize cAMP to mediate communication between cells (Sunahara, R. K., et al., Annu. Rev. Pharmaco. Toxicol., 1996, 36: 461-80; Taussig, R., et al., J. Biol. Chem., 1995, 270:1-4). For example, in mammals, signals arising from other cells such as hormones, neurotransmitters, and olfactants, modulate tmAC activity via cell surface receptors and G proteins (Taussig, R., et al., Adv. Second Messenger Phosphoprotein Res., 1998, 32:81-98). A similar cAMP signaling cascade is present in other multicellular organisms, including Drosophila (Cann, M. J. et al., Adenylyl Cyclases, 32. Lippincott-Raven, 1998; Cann, M. J. et al., Adenylyl Cyclase, 32. Lippincott-Raven, 1999; lourgenko, V., et al, FEBS Lett., 1997, 413:104-8; lourgenko, V. et al., xe2x80x9cA calcium inhibited Drosophila adenylyl cyclase (submitted); Levin, L. R., et al., Cell, 1992, 68:479-89), C. elegans (Bargmann, C. I., et al., Science, 1998, 282:2028-33; Berger, A. J., et al., J. Neurosci., 1998, 18:2871-80-; Korswagen, H. C. et al., Embo J., 1998, 17:5059-65), and Dictyostelium (Pitt, G. S. et al., Cell, 1991, 69:305-15). In contrast, the ACs found in unicellular eukaryotes and bacteria transmit nutritional information to the inside of the cell (Danchin, A., Adv. Second Messenger Phosphoprotein Res., 1991, 27:109-62).
Current models for cAMP signal transduction in mammals involve only transmembrane adenylyl cyclases (tmACs), which generate cAMP near the plasma membrane (Hempel, C. M., et al., Nature, 1996, 384:166-9; Sunahara, R. K., et al., Annu. Rev. Pharmacol. Toxicol., 1996, 36:461-80; Taussig, R., et al., J. Biol. Chem., 1995, 270:1-4). With the major effector of cAMP, the cAMP-dependent protein kinase (PKA), tethered to intracellular sites often far removed from the plasma membrane by a family of A Kinase Anchoring Proteins (AKAP) (Lester, L. B. et al., Recent Prog. Horm. Res., 1997, 52:409-29; Pawson, T., et al., Science, 1997, 278:2075-80) these models depend upon diffusion of cAMP past membrane-proximal targets to activate intracellular PKA at more distal sites. Furthermore, it must survive in a cytoplasm filled with phosphodiesterases (Beavo, J. A., et al., Mol. Pharmacol., 1994, 46:399-405; Bushnik, T., et al., Biochem. Soc. Trans., 1996, 24:1014-9). However the evidence for cAMP diffusion is based on exogenous addition of millimolar concentrations of cAMP (Bacskai, B. J., et al., Science, 1993, 260:222-6), and experiments which demonstrate diffusion of liberated PKA catalytic subunit (Bacskai, B. J., et al., Science, 1993, 260:222-6; Hempel, C. M., et al., Nature, 1996, 384:166-9). Thus there has not been a satisfactory explanation for the problems associated with how these models operate.
In addition to tmACs, another type of AC activity has been described in mammals, that of soluble adenylyl cyclase (sAC), which is thought to be expressed only in testis and sperm (Ahn, S., et al., Mol. Cell Biol., 1998, 18:967-77; Bacskai, B. J., et al., Science, 1993, 260:222-6). sAC activity appears to be biochemically and chromatographically different from tmACs, particularly a genetically engineered tmAC which is soluble, and soluble guanylyl cyclases previously described in testis (Neer, E. J., J. Biol. Chem., 1978, 253:5808-5812; Neer, E. J. et al., Biochim. Biophys. Acta, 1979, 583:531-534; Braun, T. et al., Biochim. Biophys. Acta, 1977,481:227-235). Unlike the known tmACs, sAC biochemical activity has been shown to depend on the divalent cation Mn2+ (Braun, T and Dods, R. F., Proc. Natl. Acad. Sci. USA, 1975, 72:1097-1101), sAC is insensitive to G protein regulation (Braun, T. et al., Biochim. Biophys. Acta, 1977, 481:227-235), and sAC displays approximately 10-fold lower affinity for the substrate ATP (Km approximately equal to 1 mM) (Neer, E. J., J. Biol. Chem., 1978, 253:5808-5812; Gordeladze, J. O. et al., Mol. Cell Endocrinol., 1981, 23:125-136; Braun T., Methods Enzymol., 1991, 195:130-136) than the tmACs (Km approximately equal to 100 xcexcM) (Johnson, R. A. et al., Methods Enzymol., 1994, 238:56-71). Based on these studies, this soluble form of AC was thought to be molecularly distinct from tmACs (Beltran, C. et al., Biochemistry, 1996, 35:7591-8; Berkowitz, L. A., et al., Mol. Cell Biol., 1989, 9:4272-81).
Semipurified soluble adenylyl cyclase activity is inhibited by submicromolar amounts of catechol estrogens (Braun, T., Proc. Soc. Exp. Biol. Med., 1990, 194:58-63). Braun demonstrated that the two hydroxyls of the catechol moiety were essential for the inhibitory interaction, estradiol and estrone were completely inactive. Catechols with aliphatic side chain like dopamine, L-dopa, and norepinephrine were able to inhibit sAC activity, but were 1,000 fold less potent.
Molecular evidence confirming that soluble AC represents a distinct form of adenylyl cyclase is lacking. Thus a need remains for the identification, cloning, characterization and purification of the signaling molecule having soluble adenylyl cyclase activity. There is a further need to modulate sAC activity in order to affect cell function.
Carbon dioxide (CO2) is the end product of metabolism in animals. It is normally released into the atmosphere via breathing, but is also soluble in cell membranes. CO2 combines with water in the presence of carbonic anhydrase (CA) to form carbonic acid (H2CO3) which dissociates to liberate a proton and bicarbonate ion (HCO3xe2x88x92).
CO2+H2O⇄H2CO3⇄HCO3xe2x88x92+H+CA
By itself, this reaction reaches equilibrium after about 4 minutes. However, in most biological systems, due to the ubiquitous presence of carbonic anhydrase, bicarbonate/CO2 equilibrium is reached nearly instantaneously (Johnson, L. R. Essential Medical Physiology, 1998, Phila. Lippincott-Raven).
In mammals, blood is a bicarbonate/CO2 buffer system, and the relationship between blood pH, bicarbonate and CO2 partial pressure can be described by the Henderson-Hasselbach equation:
pH=6.1+log([HCO3xe2x88x92]/0.03PCO2)
This equilibrium in serum is tightly controlled in two ways; the kidneys regulate the bicarbonate concentration and the breathing frequency determines the concentration of carbon dioxide.
Bicarbonate is the carbon source for the initial reactions of gluconeogenesis and ureagenesis (Henry, Annu. Rev. Physiol., 1996, 58:523-538). Additionally, CO2 and/or bicarbonate have been shown to modulate a number of physiological processes (i.e., diuresis, breathing, blood flow, cerebrospinal fluid formation, aqueous humor formation, and spermatocyte development). In most cases, the effects of CO2 have been ascribed to as yet undescribed chemoreceptors, and the effects of bicarbonate are usually thought to be mediated by changes in cellular pH (Johnson, 1998).
Measurement of physiological levels of bicarbonate is typically determined indirectly by calculation from the direct measurement of carbon dioxide and pH using the Henderson Hasselbalch equation. However, a certain degree of error is inherent in indirect measurements, for example, due to artifacts in arterial blood sampling. Such errors may have grave consequences to the treatment of acutely ill patients, particularly neonates, and impairs the proper diagnosis of conditions such as respiratory and metabolic acidosis or alkalosis. Indeed, current state-of-the-art portable instruments, useful in emergency or point-of-site testing, such as the i-Stat(copyright), or SenDx 100(copyright) only measure pH, CO2, and PO2 and calculate the bicarbonate levels based on these measurements. Therefore, there is a need for more accurate and direct determination of physiological bicarbonate levels.
In the yeast Saccharomyces cerevisiae, orthologs of the mammalian Ras oncogene control the cell""s physiological response to nutritional status by modulating adenylyl cyclase activity (Mybonyi, K., et al, Mol. Cell Biol., 1990, 10:4518-23; Toda, T., et al., Cell, 1985, 40:27-36; Wigler, M., et al., Cold Spring Harb. Symp. Quant. Biol., 1988, 53:649-55). Yeast AC, which is encoded by the cyrl gene, is found in a complex with Cyclase Associated Protein (CAP); this association with CAP is required for Ras-responsiveness (Field, J., et al., Cell, 1990, 61:319-27; Gerst, J., et al., Mol. Cell Biol., 1991, 11:1248-57). Interestingly, cAMP regulation by Ras proteins in S. cerevisiae is the only biochemical pathway in yeast not thought to be conserved in mammals.
In mammals, a broad family of Ras-related, small GTP-binding proteins seems to be involved in cell growth, proliferation, and differentiation (Bos, J. L., et al., EMBO J., 1998, 17:6776-82). Ras genes are potent oncogenes; they are thought to be mutated in 30% of all human tumors. Although the mechanism of Ras transformation in human tumorigenesis has been the focus of intense research over the past years (Chang, E. H., et al., Nature, 1982, 297:479-83; Der, C. J., et al., Proc. Natl. Acad. Sci. USA 79:3637-40; Goldfarb, M., et al., Nature, 1982, 296:404-9; Parada, L. F., et al., Nature, 1982, 297:474-8), its mechanism of oncogenic transformation is still not completely understood. A protein kinase cascade involving Raf protein kinase activation of the MAPKinase cascade is downstream from Ras, as are activation of PI-3 kinase and the Ras-family member Ral (Gille, H., et al., J. Biol. Chem., 1999, 274:22033-40; Osada, M., et al., Mol. Cell Biol., 1999, 19:6333-44; Rosario, M., et al., Embo. J., 1999, 18:1270-9). However, these do not tell the entire story; a number of other genes have been proposed to play a role in Ras transformation (Tang, Y., et al., Mol. Cell Biol., 1999, 19:1881-91). Among the best characterized Ras effectors, constitutively active forms of Raf or the various MAPKinase can transform fibroblasts on their own, suggesting this kinase cascade can mediate at least part of the transforming functions of Ras. But oncogenic forms of these kinases are not as efficient at transforming fibroblasts as oncogenic forms of Ras, and inhibition of MAPKinase activity does not completely block transformation of Ras (Denouel-Galy, A., et al., Curr. Biol., 1998, 8:46-55; Yip-Schneider, M. T., et al., Int. J. Oncol., 1999, 15:271-9). In contrast, constitutive activation of PI-3 kinase does not transform cells, but inhibiting its activity is sufficient to prevent transformation by oncogenic Ras (Rodriquez-Viciana, P., et al., Cell, 1997, 89:457-67). Together, these data suggest that signaling through PI-3 kinase is necessary but not sufficient for oncogenic transformation by Ras proteins, while the MAPKinase pathway is at least partially sufficient, but does not appear to be necessary. Thus, there is a need to identify other components of Ras mediated transformation.
The present invention provides soluble adenylyl cyclase (sAC), a signaling enzyme which produces cAMP in eukaryotic, particularly non-yeast eukaryotic cells. The natural soluble form of adenylyl cyclase generates cAMP at a distance from the membrane, and thus closer to its required site of action. Accordingly, soluble adenylyl cyclase is useful in regulating or controlling cAMP production.
In a first aspect, the present invention provides an isolated nucleic acid molecule encoding a soluble adenylyl cyclase. The nucleic acid molecule is selected from the group consisting of a nucleic acid which encodes a polypeptide having an amino acid sequence as set out in SEQ ID NO. 1 or SEQ ID NO: 11, a splice variant thereof or an allelic variant thereof. Alternatively, the invention provides a nucleic acid molecule which hybridizes under stringent conditions to the nucleic acid sequence set out in SEQ ID NO: 2 or SEQ ID NO: 12. A nucleic acid molecule having at least a twenty nucleotides sequence identical to a corresponding twenty nucleotide sequence as set out in SEQ ID NO: 2 or SEQ ID NO: 12 is also encompassed. Yet a further alternative nucleic acid sequence encodes a soluble polypeptide having an amino acid sequence sufficiently duplicative of the soluble adenylyl cyclase encoded by SEQ ID NO: 1 or SEQ ID NO: 11 so that a polypeptide expressed from the nucleic acid molecule has the biological property of catalyzing the production of cyclic AMP, which polypeptide has a catalytic domain having a sequence that is not more than 16% similar to a catalytic domain of a mammalian transmembane adenylyl cyclase as determined by CLUSTAL analysis.
In one embodiment the soluble adenylyl cyclase is mammalian soluble adenylyl cyclase. In a preferred embodiment the adenylyl cyclase is human soluble adenylyl cyclase; rat soluble adenylyl cyclase is also provided.
The invention further provides a vector comprising the nucleic acid molecule as defined above. The vector can be an expression vector, in which the nucleotide sequence encoding soluble adenylyl cyclase is operably associated with an expression control sequence, e.g., for expression in human cells.
The invention further provides a host cell, preferably a mammalian cell, which comprises said expression vector.
The present invention also provides a method for producing recombinant soluble adenylyl cyclase comprising isolating soluble adenylyl cyclase expressed by a host cell containing an expression vector encoding a soluble adenylyl cyclase. In one embodiment, the soluble adenylyl cyclase is isolated using an anti-soluble adenylyl cyclase antibody.
The discovery of soluble adenylyl cyclase provides a mechanism for screening factors which modulate soluble adenylyl cyclase induced signaling. Thus the present invention provides a method of screening for a modulator of soluble adenylyl cyclase-induced signaling, which method comprises detecting inhibition of a signal of a soluble adenylyl cyclase-induced signal transduction pathway in a cell in the presence of a candidate compound wherein detection of inhibition of the signal indicates that the candidate compound is an inhibitor of soluble adenylyl cyclase-induced signaling. In one embodiment of the invention, the signal is cAMP generation. In this manner, cell proliferation, cell differentiation and apoptosis, control of which are diminished in pathological conditions such as cancer, can be modulated. Such modulation may have therapeutic and prophylactic benefit for those subjects suffering from such pathological conditions. Current treatment for these conditions, including chemotherapy and radiation therapy, are typically nonspecific and often have deleterious side effects. The identification of novel agents directed to a specific target to treat such conditions would be greatly advantageous.
The present invention also provides a method of modulating cAMP production by modulating the expression of soluble adenylyl cyclase. In this manner, for example, aberrant cell proliferation can be decreased or inhibited by regulating or modulating cAMP production.
With respect to decreasing cell proliferation, the present invention provides a method for decreasing or inhibiting soluble adenylyl cyclase expression such that cAMP production is decreased or inhibited and cell proliferation is decreased or inhibited.
Accordingly the present invention provides a method of regulating certain medical or pathological conditions in which cAMP production is implicated.
The present invention further provides a method of identifying factors which inhibit soluble adenylyl cyclase activity either by blocking expression of the soluble adenylyl cyclase gene or down regulating its ability to regulate cAMP metabolism. Such factors can thus be used in vivo or in vitro to regulate soluble adenylyl cyclase activity.
The present invention also provides a method for modulating soluble adenylyl activity by increasing soluble adenylyl cyclase activity. Soluble adenylyl cyclase can be activated or its activity can be increased when stimulation of cAMP production is desired. The present invention further provides a method of identifying factors that stimulate or enhance soluble adenylyl cyclase activity.
Applicants have specifically found that soluble adenylyl cyclase is potently stimulated by sodium bicarbonate. Controlling intracellular or extracellular bicarbonate concentrations provides an additional mechanism through which soluble adenylyl cyclase activity, and ensuing cAMP production, can be regulated.
In this regard, applicants have found that regulating cAMP concentrations by modulating soluble adenylyl cyclase activity can provide a means of modulating sperm capacitation. The present invention provides a method of regulating sperm capacitation by regulating expression of soluble adenylyl cyclase. In one embodiment, the invention provides a method for reducing or inhibiting male germ cell fertility by reducing or inhibiting soluble adenylyl cyclase activity and thereby inhibiting or decreasing capacitation of sperm. In one aspect the method comprises administering to a subject an agent that inhibits soluble adenylyl cyclase activity in an amount effective to decrease or inhibit cAMP production. In this manner, the fertilization of an ovum can be inhibited.
In another embodiment, the invention provides a method for increasing or enhancing the ability of sperm to fertilize an egg. With respect to this embodiment, the present invention provides a method of increasing sAC activity by, for example, treating sperm with a small molecule agonist, or using gene therapy to stimulate or enhance sperm capacitation. In this manner, the likelihood of fertilization of an ovum is increased or enhanced. This is particularly useful in procedures such as in vitro fertilization.
Applicants have also found that regulating cAMP concentrations by regulating soluble adenylyl cyclase expression provides a means of regulating insulin secretion of pancreatic islet cells. cAMP is needed for the release of insulin (Liang Y et al., Annual Review of Nutrition, 1994, 14:59-81). Addition of bicarbonate activates soluble adenylyl cyclase to increase cAMP production. With the discovery and isolation of soluble adenylyl cyclase, applicants provide a novel means of increasing or stimulating insulin release from the pancreas, when normal physiological mechanisms fail to do so, by increasing soluble adenylyl cyclase activity.
Thus, the invention provides a method of increasing insulin secretion of pancreatic islet cells comprising increasing soluble adenylyl cyclase activity.
The present invention further provides a method of treating glaucoma by reducing aqueous humor formation. With respect to this embodiment, aqueous humor formation, which is stimulated by cAMP signaling, can be reduced or inhibited by administering to a subject afflicted with glaucoma a modulator of sAC activity in an amount effective to reduce or inhibit bicarbonate dependent sAC activity to decrease the production of cAMP.
Applicants have also discovered that bicarbonate activates sAC in a direct, specific and pH independent manner. Accordingly, the present invention provides a method of quantifying bicarbonate in a body fluid using soluble adenylyl cyclase. The body fluid can be blood, urine, aqueous humor and the like.
In one aspect, the method of quantifying comprises contacting the body fluid with sAC. Contact of bicarbonate with sAC activates sAC and generates cAMP. The amount of bicarbonate in the sample can be correlated to the amount of cAMP detected. Measurement or detection of cAMP can be effected through a number of means including fluorescence, colorimetry and the like. Due to its specificity for sAC, a direct correlation to the amount of bicarbonate in the sample body fluid can be made using this method.
Applicants have further discovered that isolated soluble adenylyl cyclase is an oncogene. The isolated soluble adenylyl cyclase of the present invention is able to transform fibroblasts in vitro leading to loss of contact inhibition. Isolated soluble adenylyl cyclase and a truncated form of soluble adenylyl cyclase (sAC), transfected into cells transformed NIH3T3 cells. Soluble adenylyl cyclase was further demonstrated to support anchorage independent growth in soft agar. The Ras related protein, Rap1 which is a specific competitive inhibitor of Ras, inhibited soluble adenylyl cyclase transformation of cells. Thus soluble adenylyl cyclase provides a target for Ras family (or other small GTPases) regulation of cAMP metabolism in mammals. This discovery is surprising inasmuch that stimulation of transmembrane adenylyl cyclase activity blocks transformation of fibroblasts by oncogenic Ras (Chen, J., et al., Science, 1994, 263:1278-81; Smit, M. J. et al., Proc,. Natl. Acad. Sci. USA, 1998, 95:15084-9).
Thus, the invention provides a method of inhibiting unwanted cell proliferation in an animal, a mammal, a human by administering an effective amount of a soluble adenylyl cyclase binding protein peptide fragment wherein the protein inhibits soluble adenylyl cyclase expression. In another aspect, the functional activity of soluble adenylyl cyclase can be inhibited by administering a specific soluble adenylyl cyclase antisense molecule to cells that express functional soluble adenylyl cyclase.
The observation that soluble adenylyl cyclase is expressed in tumor cells further provides a diagnostic marker to detect the presence of pathological conditions in an animal, e.g., a mammal. In accordance with this aspect, the present invention provides a method of diagnosing the onset of, or the likelihood of onset of, or for monitoring the course and severity of a pathological condition derived from soluble-adenylyl cyclase activation, comprising detecting an increase in soluble adenylyl cyclase levels in a biological sample obtained from a subject suspected of suffering from such conditions.
The invention further provides compositions and kits for the diagnosis of conditions arising from soluble-adenylyl cyclase activation.
These and other aspects of the invention are more fully set forth in the Drawings, Detailed Description, and Examples.